Plastics are inexpensive materials capable of realizing a diverse range of properties corresponding to the structure thereof, thereby making them indispensable for modern society. However, nearly all plastics are synthesized from petroleum raw materials, and have been developed and produced with the objective of achieving long-term stability. As a result, since the majority of petroleum-based synthetic plastics are not degraded in the natural environment following their disposal, the management and disposal of used plastic waste is becoming a major problem in countries throughout the world. In addition, the depletion of fossil fuels, as represented by petroleum, is also becoming a serious problem. Even if the duration during which fossil fuel resources can be recovered is able to be extended through advances made in the field of mining technology, these resources are still finite, and the consumption of fossil fuel resources is predicted to continue to increase in the future due to increased demand stemming from global economic growth. What is more, rising carbon dioxide levels in the air attributable to consumption of fossil fuel resources is also becoming a significant environmental problem. Consequently, it is necessary to curtail consumption of fossil fuel resources and establish a social structure that is not dependent on fossil fuel resources.
In view of these circumstances, there is a desire to develop and practically implement the use of plastics that reduce the burden on the environment in the form of bioplastics. Bioplastics is the generic term for two types of plastics consisting of “biodegradable plastics” and “biomass plastics”. Biodegradable plastics are plastics that are degraded by microorganisms in the environment and do not cause problems in terms of waste disposal as is the case with petroleum-based synthetic plastics. In addition, biomass plastics refer to plastics that use renewable biomass resources derived from plants and animals used as raw materials, and since the carbon dioxide generated by their combustion is carbon dioxide that would have been inherently present in air following the fixation thereof by plant photosynthesis (making it carbon neutral), it can be said to be material capable of contributing to the future establishment of a recycling society from the viewpoints of depletion of fossil fuel resources and discharge of greenhouse gases.
Polyhydroxyalkanoates (PHA), which are accumulated in the cells of numerous microorganisms for use as an energy source, are expected to serve as plastic materials synthesized from biomass as a raw material and is biodegradable. Although poly(3-hydroxybuturate) (P(3HB)) is a typical example of a PHA biosynthesized by various microorganisms, since P(3HB) has the properties of being hard and brittle, its practical application as a material is difficult. On the other hand, since many PHA copolymers, which are hydroxyalkanoates having different structures as comonomer units, have improved flexibility and other properties, many researches have been actively conducted on the biosynthesis of PHA copolymers. For example, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer is biosynthesized by providing a precursor in the form of propionic acid or pentanoic acid, while poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer is biosynthesized by adding 1,4-butandiol or γ-butyrolactone. More recently, research is proceeding in several countries on the biosynthesis of PHA copolymers by genetically modified microorganisms from biomass in the absence of the addition of precursor.
Poly((R)-3-hydroxybutyrate-co-3-hydroxyhexanoate) copolymer (P(3HB-co-3HHx)), which is biosynthesized by some bacteria such as a soil bacterium Aeromonas caviae using vegetable oil or fatty acids as carbon sources, demonstrates an increase in flexibility corresponding to the polymer composition thereof, and a polymer containing a (R)-3-hydroxyhexanoate (3HHx) unit at about 10 mol % has been determined to be a plastic that demonstrates suitably superior flexibility. The fraction of 3HHx of P(3HB-co-3HHx) biosynthesized by A. caviae from vegetable oil is present at 10 mol % to 20 mol %, and although this plastic demonstrates flexibility suitable for practical applications, the accumulation thereof in bacterial cells is low at only about 15% by weight, thereby making its application to actual production difficult (see Patent Document 1, Patent Document 2 and Non-Patent Document 1).
Therefore, a recombinant strain that efficiently produces P(3HB-co-3HHx) having a high fraction of 3HHx using vegetable oil as a carbon source was developed by allowing a PHA polymerase having broad substrate specificity to function in Cupriavidus necator, which is known to efficiently produce P(3HB), and then modifying the polyester biosynthesis pathway and β-oxidation pathway that decomposes fatty acids (see Patent Document 3, Patent Document 4, Patent Document 5, Patent Document 6, Non-Patent Document 2, Non-Patent Document 3, Non-Patent Document 4 and Non-Patent Document 5).
In the aforementioned research on the microbial synthesis of P(3HB-co-3HHx), long-chain fatty acids, butyric acid or butanol was used as raw materials. On the other hand, technology for the production of P(3HB-co-3HHx) using a saccharide derived from a polysaccharides as raw materials would be extremely important when considering that polysaccharides constitute a reproducible biomass resource that is present in large amounts on the earth. However, although the only reported wild-type strain of microorganism capable of biosynthesizing P(3HB-co-3HHx) from a saccharide raw material is Bacillus sp. strain INT005, which produces P(3HB-co-3HHx) from glucose, the fraction of 3HHx is low at 1.6 mol % and its biosynthesis pathway has yet to be unclear (see Non-Patent Document 6).
There are also extremely few reports on the biosynthesis of P(3HB-co-3HHx) from saccharide raw materials by recombinant microorganisms. Qiu et al. reported biosynthesis of P(3HB-co-19 mol % 3HHx) at 10% by weight by a recombinant strain of Pseudomonas putida possessing an enzyme which produces (R)-3-hydroxyhexanoyl-CoA ((R)-3HHx-CoA) from an intermediate in fatty acid biosynthesis pathway by transthioesterification, along with (R)-3-hydroxybutyryl-CoA ((R)-3HB-CoA)-generation pathway. They also reported biosynthesis of P(3HB-co-14 mol % 3HHx) at 10% by weight by a recombinant strain of Aeromonas hydrophila expressing thioesterase capable of releasing an long acyl group by cleaving acyl-acyl carrier protein intermediates in fatty acid biosynthesis pathway followed by generation of (R)-3HHx-CoA via decomposition of long acyl group thereof (see Non-Patent Document 7).
On the other hand, the inventors of the present invention previously examined biosynthesis of P(3HB-co-3HHx) from fructose raw material by a recombinant strain of C. necator. Namely, a pathway was designed to form butyryl-CoA from an intermediate having four carbon atoms formed by condensing two molecules of acetyl-CoA derived from a saccharide, and to generate an intermediate having six carbon atoms by condensing the butyryl-CoA with another molecule of acetyl-CoA, and then to copolymerize (R)-3HHx-CoA produced by further conversion of the six carbon intermediate and (R)-3HB-CoA. In order to establish this artificial metabolic pathway, (R)-specific enoyl-CoA hydratase gene phaJAc, derived from A. caviae producing crotonyl-CoA from (R)-3HB-CoA, and crotonyl-CoA reductase gene ccrSc derived from an actinomycete Streptomyces cinnamonensis, which reduces the double bond of crotonyl-CoA, were introduced into a PHA polymerase-deficient strain PHB−4 along with PHA polymerase gene phaCAc having broad substrate specificity. As a result, the resulting recombinant strain biosynthesized P(3HB-co-3HHx) containing 1.2 mol % to 1.6 mol % of the 3HHx unit from fructose as the only carbon source, and although the designed pathway was certainly found to be functional, the fraction of 3HHx was too low to improve the polymer properties (Non-Patent Document 8).